The effect of various heparin concentrations on the radioimmunoassay of gentamicin was studied in vitro and in rats. Heparainized test tubes were prepared, and whole blood was added followed by a gentamicin sulfate solution. Eight Sprague-Dawley rats were given heparin or gentamicin or both via infusion, and blood samples were drawn and tested. A radioimmunoassay for gentamicin was used in both the in vitro and in vivo tests. When 0-1000 units/ml of heparin were added to a constant amount of gentamicin (12 micro grams) in vitro, no difference was found in the assayed gentamicin concentration (p greater than 0.01). Similarly, when gentamicin concentrations of 0-16 micro grams /ml were added to a constant amount of heparin (20 units), the assays yielded results similar to the known gentamicin concentrations added. No significant difference was found in the clearance rate (p greater than 0.05) or terminal half-lives (p greater than 0.05) of gentamicin in treated and nontreated rats. The results suggest that variable heparin concentrations do not affect gentamicin concentrations as determined by radioimmunoassay.